Throughout this application various publications are referenced, many in parenthesis. Full citations for each of these publications are provided at the end of the Detailed Description. The disclosures of each of these publications in their entireties are hereby incorporated by reference in this application.
Despite the promise and excitement of gene therapy, it is still a long way from practice. Two major problems hindering gene therapy are that (1) gene transfers to non-dividing cells are still extremely inefficient, and (2) gene transfer to specific desired non-dividing cells within a population of other cell types is even more inefficient. Thus, any way to increase the amount of gene transfer will greatly benefit this emerging field.
Many techniques and vectors for gene therapy have been developed to target genes to cells, including replication-deficient recombinant retroviruses, adenoviruses, and adeno-associated viruses, as well as non-viral vectors such as ligand-DNA conjugates or DNA lipofection. However, most targeting techniques developed to date have only addressed the ability to internalize the DNA into the cytoplasm of the cell. It is clear that gene therapy relies on the ability of targeted genes to enter the nucleus. This is true regardless of how the DNA or RNA is targeted to the cell; once within the cytoplasm, the gene must become nuclear to be transcribed, replicated, and maintained either in an integrated or episomal state, yet there has been little attention directed toward either discovering or exploiting the mechanisms used by the cell to direct DNA to the nucleus.
Recent work has begun to address the nuclear targeting and entry of plasmid DNA. Using transformed cell lines and primary cultured cells, it has been shown that plasmid DNA is able to enter the nuclei of cells in the absence of cell division and its accompanying nuclear envelope breakdown (Dean 1997). As with all other macromolecular exchange between the cytoplasm and nucleus (for a review, see Nigg 1997), DNA nuclear entry appears to be mediated by the nuclear pore complex (Dean 1997; Dowty et al. 1995). A 366 bp sequence of DNA containing the simian virus 40 (SV40) origin of replication and early promoter has been identified that is absolutely necessary for the nuclear entry of plasmid DNA in cultured cell lines derived from monkey, rat, mouse, hamster, and human origin, as well as non-transformed primary cells from rat, chicken, and human tissues (Dean 1997). Thus, nuclear import of plasmid DNA is signal-dependent and occurs in all eukaryotic cells tested to date. This DNA nuclear localization signal has been further localized to regions within the 366 bp DNA fragment. See also PCT International Publication No. WO 97/34915, published September 25, 1997, by Dean (the contents of which are hereby incorporated by reference). These results demonstrate that transport of DNA into the nucleus is sequence-specific.
The promise and potential of gene therapy techniques to cure or to alleviate symptoms in a multitude of disorders and diseases results in a continuing need for ways to increase the amount of gene transfer to cells. Ideally, a way to increase the amount of gene transfer to a specific cell type would exist for cell-specific targeting of gene therapy.